SUMMARY

How do honeybees use visual odometry and goal-defining landmarks to guide
food search? In one experiment, bees were trained to forage in an
optic-flow-rich tunnel with a landmark positioned directly above the feeder.
Subsequent food-search tests indicated that bees searched much more accurately
when both odometric and landmark cues were available than when only odometry
was available. When the two cue sources were set in conflict, by shifting the
position of the landmark in the tunnel during test, bees overwhelmingly used
landmark cues rather than odometry. In another experiment, odometric cues were
removed by training and testing in axially striped tunnels. The data show that
bees did not weight landmarks as highly as when odometric cues were available,
tending to search in the vicinity of the landmark for shorter periods. A third
experiment, in which bees were trained with odometry but without a landmark,
showed that a novel landmark placed anywhere in the tunnel during testing
prevented bees from searching beyond the landmark location. Two further
experiments, involving training bees to relatively longer distances with a
goal-defining landmark, produced similar results to the initial experiment.
One caveat was that, with the removal of the familiar landmark, bees tended to
overshoot the training location, relative to the case where bees were trained
without a landmark. Taken together, the results suggest that bees assign
appropriate significance to odometric and landmark cues in a more flexible and
dynamic way than previously envisaged.

Introduction

Honeybees Apis mellifera use a combination of sensory cues to
guide navigation (von Frisch,
1993). These include both long-range (e.g. odometry, compass
direction) and short-range cues (e.g. scent, landmark). An unresolved problem
is how bees integrate these sensory cues to guide their return to various
places in the world, such as the location of a food source. It is often
assumed, for instance, that long-range cues are sufficient to guide a bee to
the general vicinity of a goal site (e.g.
Collett and Collett, 2002),
and that landmark cues are used to pinpoint the exact goal location
(Cartwright and Collett, 1983).
However, few experiments have explored this latter hypothesis in a systematic
fashion (cf. Chittka et al.,
1995a). This study examines the functional roles and interactions
of visual odometry (the distance sense of the bee) and landmark guidance
during food search, within the context of a scaled-down foraging
environment.

Until recently, it was believed that honeybees use the amount of energy
expended on a given flight as an index of distance travelled
(von Frisch, 1993).
Accumulating evidence now suggests that honeybees use visual information to
measure how far they have flown in a particular direction
(Cheng et al., 1999;
Chittka and Tautz, 2003;
Esch and Burns, 1996;
Esch et al., 2001;
Si et al., 2003; Srinivasan et
al., 1996,
1997,
1998,
1999,
2000;
Tautz et al., 2004). In
particular, distance appears to be measured in terms of the amount of optic
flow, or visual motion, that occurs on the eye during a given flight (i.e.
integrated optic flow). For example, honeybees trained to forage in an
environment rich in optic flow, such as a narrow tunnel lined with a textured
pattern, dramatically overestimate the actual distance flown, as indicated by
their dance behaviour (Esch et al.,
2001; Si et al.,
2003; Srinivasan et al.,
2000). However, no comparable overestimation occurs when bees fly
through an environment impoverished in optic flow, namely, a tunnel lined with
stripes oriented along the direction of travel
(Si et al., 2003;
Srinivasan et al., 2000).
Srinivasan et al. (1996,
1997,
1998) showed that honeybees
can use visual odometry to guide food search, independently of cues such as
visual landmarks, scent, time of flight and energy expenditure. Perhaps the
most compelling evidence that bees use integrated optic flow to guide search
comes from a control experiment, in which bees were trained and tested in
tunnels lined with axially oriented stripes, such as those used in the dance
experiments of Srinivasan et al.
(2000). In this situation,
bees simply flew from one end of the tunnel to the other during their search,
indicating that they could not locate the training position with any accuracy
in the absence of optic flow.

In the present study, bees were trained to forage in textured tunnels, such
as those used by Srinivasan et al.
(1996,
1997,
1998), with a visual landmark
directly above the reward site. By changing the position of the landmark in
the test conditions, and the availability of odometric cues during training
and test, an attempt was made to tease apart the relative contributions of
odometry and landmark cues to navigation and search behaviour. We devised
various experiments to address the following questions. Experiment 1: Does the
presence of a learned landmark increase the accuracy of search behaviour,
relative to the situation in which odometry alone guides search? Do odometry
or landmark cues predominate when the two sets of cues are made to conflict,
for example, by shifting the position of the landmark at test? Experiment 2:
What is the effect of depriving the bees of visual odometry while allowing the
use of landmark cues? Experiment 3: Is it necessary that the landmark be
present during training (i.e. learned), or do bees use any landmark cues near
the goal to guide search? Experiments 4 and 5: Does the tunnel distance to
which bees are trained significantly affect the relative significance of
odometry and landmark cues?

Materials and methods

Location and equipment

Experiments were conducted outdoors in a relatively open area in the
Wallaby Compound of The Australian National University, unless otherwise
specified (i.e. Experiment 5), using tunnels either 3.4 m or 7.8 m
(Experiments 4 and 5) long, 0.2 m high and 0.22 m wide. The side of each
tunnel was marked every 0.2 m, enabling quantification of search patterns. The
entire tunnel was covered with either Plexiglass sheets or (on hot days) nylon
mesh, to prevent bees from entering or exiting at any location other than
through the tunnel.

The landmark was a piece of rigid white cardboard placed on the top of the
tunnel, spanning one lateral wall to the other, and encompassing the whole of
the 0.2 m unit on which it was placed. Since the landmark spanned the entire
unit, bees could make several ∪-turns within this segment before finally
crossing over into an adjacent segment. In this sense then, the recording
criterion underestimated the number of ∪-turns made by bees anywhere in
the tunnel, but especially at the landmark site. The landmark being large and
dorsal, however, had the advantage of obscuring the bees' views of any
external landmarks (e.g. branches of distant trees).

Training

Italian honeybees Apis mellifera L. from a single colony were
trained to forage at a feeder located at a specified position within a tunnel
for a full day (8 h training) before testing began. Bees flew from the hive to
the tunnel, located around 50 m away. The feeder was a small plastic container
(100 ml capacity), with a flat circular-shaped base through which bees could
extract small amounts of sucrose solution. The sucrose concentration was 1 mol
l-1 at the start of training but was modulated slightly throughout
the experiment to keep an approximately constant number of bees coming to the
experiment. The tunnel was lined with paper printed with random black and
white 1 cm2 texture elements. In each experiment, approximately 20
bees were marked individually with coloured paint and trained to locate the
food reward in the training tunnel. We ensured that nearby landmarks were not
visible from the bees' vantage point in the tunnel.

Food search

Bees were tested in the training tunnel in Experiments 2, 4 and 5. In
Experiments 1 and 3, bees were tested in a tunnel in which the feeder was
periodically placed at a random location. To accomplish this, the training
protocol was interrupted hourly for a period of 5-10 min, during which the
training bees foraged in the testing tunnel. At test, individual bees flew
through the tunnel towards the position previously occupied by the feeder. At
some point during a given flight, the bee began to search for the missing
feeder, performing a series of ∪-turns, each time reversing its direction
of travel in the tunnel (Fig.
1). Search flights were quantified by observing the first four∪
-turns conducted by each bee upon entering the tunnel. A ∪-turn was
defined as a crossing-over between adjacent units in the tunnel (e.g. from 9
to 8), and was recorded manually on paper by the experimenter.

Schematic illustration of the training and testing set-up. (A) Bees were
trained to forage in a textured tunnel (the specific texture was varied
throughout experiments) with a feeder at a designated location, and the
landmark directly above. (B) In the test situation, bees entered the tunnel
individually, and began searching for the removed feeder, repeatedly
traversing the tunnel. The cross-section shows the trajectory over four∪
-turns (note this is not a space-time diagram in the strict sense).

Experiment 1

Bees were trained to forage at a feeder placed in a tunnel lined with a
randomly textured pattern, such as that used by Srinivasan et al.
(1997). A conspicuous visual
landmark was placed directly above the location of the feeder. In the test
situation, the feeder was removed and bees' search patterns were assessed (a)
when the landmark was removed altogether, (b) with the landmark in place at
the training position, or (c) with the landmark displaced relative to the
training location, thereby setting up a situation in which odometry and
landmark cues were in positional conflict.

Experiment 2

Experiment 2 was similar to Experiment 1 except that the training tunnel
was lined with parallel stripes oriented along the main axis of the tunnel.
Since such axial stripes do not produce a significant image motion on the eye,
bees cannot gauge distance travelled (Si
et al., 2003; Srinivasan et al.,
1997,
2000). In the present
instance, this would apply equally to measurements made either relative to the
tunnel entrance or relative to the goal-defining landmark. Experiment 2
thereby assessed what kind of search strategies bees adopt when only landmark
cues are available to locate the feeder.

Experiment 3

Bees were trained in a tunnel lined with random texture, but without a
landmark at the feeder location. In the test conditions, a landmark was placed
at one of several locations in the tunnel to examine how the addition of novel
landmark cues affects search behaviour. An additional control condition
assessed how bees searched when tested without the novel landmark.

Experiments 4 and 5

Bees were trained to a longer distance than in Experiment 1 in order to
examine whether search behaviour would differ from that observed at shorter
distances.

Data analyses

The order in which conditions were tested was randomised within blocks,
each block testing all conditions; once tested, a condition was excluded until
all had been tested. Each block was tested at least twice. For each condition,
search distributions were calculated on the basis of the first two∪
-turns. These ∪-turns typically provided sufficient information to
analyse search behaviour (e.g. Cheng et
al., 1999). In cases where the third and fourth turns illustrated
important aspects of the bees' navigation strategies, these data were also
analysed. The search distribution of a group of bees was calculated for each
test condition, as follows. For each flight, all tunnel units between the
positions of first and second ∪-turns were assigned values of one. Each of
these values was then divided, or weighted, by the total path length between
the first and second ∪-turns (inclusive). These weighted scores were then
summed, for each tunnel unit, across all the flights in an experimental
condition, and divided by the total number of flights. Thus, the total area
under the curve representing the search distribution was normalized to one.
Due to the normalization with respect to path length (i.e. distance from first
to second ∪-turns), each flight segment contributed the same area to the
curve. That is, shorter path lengths (associated with the more accurate
searches) contributed the same bulk to the search distribution as longer path
lengths. However, shorter (more accurate) path lengths contributed more to the
height of the search distribution, because the value associated with each
tunnel unit was higher.

All figures also show the positions of first and second ∪-turns
normalized to the total number of flights, giving the relative frequency of∪
-turns across all units. When analysing only the first two ∪-turns
the flight path-segment between first and second ∪-turns for each
individual flight were displayed graphically, which supplemented the histogram
representations of ∪-turns (which do not give information about individual
flight paths). Indeed, displaying individual flight paths makes it immediately
possible to visualise the link between ∪-turn position and the search
distribution.

Statistics

Statistical analyses (analysis of variance, ANOVA) were conducted for each
experiment on the first and second ∪-turn data, and in appropriate cases,
on the third and fourth ∪-turn data. These analyses indicated whether the
position of the landmark at test had an overall effect on the means of∪
-turns 1 and 2 across conditions. The results of these en bloc
statistical analyses are stated only briefly in the text; details can be found
in the Appendix. In special instances, where a comparison between similar
conditions in different experiments was of particular importance, individual
statistical tests were undertaken as stated in the text. Analyses were
performed using Matlab software, Version 6.1 (MathWorks, Inc.) and Genstat for
Windows, Release 6.1 (USN International, Ltd).

Results

Experiment 1

The data from this experiment are shown in
Fig. 2. The positions of first
and second ∪-turns are plotted as frequency histograms (normalized to
flight number), and the search distribution, calculated as described in
Materials and methods, is overlaid on top. The path segments between the first
and second ∪-turns, for each flight, are plotted above the histograms and
search distributions, showing the positions of first and second ∪-turns,
and for each flight, the line joining these positions indicates the length of
the path segment.

Results of Experiment 1. (A) Bees tested with no landmark conducted a broad
search. (B) With the landmark at the training location, bees searched very
accurately. (C) When the landmark was shifted to unit 14, bees searched either
at the landmark or the training location. (D) Bees searched at the landmark
when it was shifted to unit 4. Black bars, first ∪-turns; grey bars,
second ∪-turns; coloured lines, search distributions; inverted triangles,
training location; diamonds, landmark position; N=flight number. Note
different y-axis scales in A,C and B,D.

The data show clearly that the presence and position of the landmark had a
dramatic effect on where bees searched. An ANOVA revealed a significant effect
of landmark position on the mean (F3,166=59.78,
P<0.001) of search. In the absence of the landmark
(Fig. 2A), bees searched very
broadly for the food, whereas search was very accurate with the landmark in
place at the training location (Fig.
2B). When the landmark was shifted towards the tunnel end
(Fig. 2C) or entrance
(Fig. 2D), bees generally
searched near the position of the landmark, rather than at the training
distance, meaning that landmark cues tended to override odometry. The overall
difference between conditions, in terms of both mean search position and
spread of search, was highly significant. The individual conditions are
examined further below.

No landmark

The search distribution in this condition shows a very broad peak in the
general vicinity of the training location
(Fig. 2A). Indeed, the search
distribution appears much broader than those previously obtained with bees
trained to the same tunnel location (e.g.
Srinivasan et al., 1997). As
indicated by the pattern of first ∪-turns, the errors were typically in
the direction of overshooting rather than undershooting the training location.
It is therefore possible that these bees were seeking the missing landmark.
Interestingly, the pattern of individual flight path segments shows that, in
many instances, bees did not pass over the training location on the transition
from first to second ∪-turns.

Landmark at unit 9

With the landmark present at the training position, bees searched almost
exclusively at this location (Fig.
2B). Essentially, bees never performed ∪-turns away from the
immediate vicinity of the landmark. This result therefore confirms the
hypothesis that landmark cues can significantly improve search accuracy,
relative to the case where only odometric cues are available (see above).

Landmark at unit 14

A more complex pattern of results emerged when the landmark was positioned
at unit 14 (Fig. 2C). On most
flights, bees searched at the position of the landmark, but there was a small
group of flights in which bees searched in the vicinity of the training
position. This division of behavioural outcomes most likely arose because, in
many cases, bees overshot the training location and subsequently sighted the
landmark. Once acquired, bees did not often disengage visually with the
landmark, as shown by the pattern of second ∪-turns. By comparing the
pattern of first ∪-turns in the current condition with that obtained in
the `No landmark' condition, it is possible to deduce the distance at which
bees first detected the landmark. This comparison therefore quantifies the
extent to which the landmark acted as a beacon (e.g.
Chittka et al., 1995a;
Collett and Rees, 1997).

Comparison between the `Landmark at unit 14' and `No landmark' conditions
reveals the point at which bees were drawn towards the landmark (i.e. a beacon
effect). (A) Reproduction of first ∪-turn distributions. (B) Cumulative
frequencies were tested statistically, and found to be different at unit 11
(see text), meaning that bees were drawn to the landmark from this point
onwards.

Fig. 3 plots the first∪
-turns for both conditions, along with the cumulative distributions
obtained by summing (and normalizing to the total number of ∪-turns) the
number of ∪-turns performed at, or before, each unit in the tunnel. These
cumulative distributions differ overall (Kolmogorov-Smirnov test,
P=0.0187). Pairwise comparisons at different tunnel units show that
the two cumulative distributions differ statistically at unit 11 (two-tailed
Fisher exact test, P<0.05) and at unit 12 (two-tailed Fisher exact
test, P<0.01), meaning bees began to detect the landmark from at
least 0.6 m away. The cumulative distributions diverge slightly earlier than
unit 11 (though the differences are not significant), indicating that some
bees may have detected the landmark before reaching unit 11.

Landmark at unit 4

When the landmark was shifted to unit 4, bees searched almost exclusively
at this location (Fig. 2D). On
a few flights (8/43), bees went past the landmark on first ∪-turns but
there is insufficient data to conclude whether these bees were searching in
accord with odometry or were simply lost. What is clear is that on most
flights bees preferred to search in accord with the landmark cue. The pattern
of results is therefore very similar to the `Landmark at unit 9'
condition.

Experiment 2

The rationale of the following experiment was to eliminate the search
component driven by odometry, thereby isolating the mechanism that depends
only on landmark cues. To this end, bees were trained at unit 9 in a tunnel
lined with black and white stripes oriented along the tunnel axis (axially
striped tunnel). The experimental protocol was the same as in Experiment 1,
except that bees were tested in the training tunnel.

We found that the position of the landmark cue again had a strong effect on
search behaviour in terms of the mean (F3,152=55.16,
P<0.001) search position. In the absence of landmark cues, the
search distribution appears approximately flat
(Fig. 4A), and as the pattern
of first and second ∪-turns shows, this distribution was due to the
tendency for bees to fly from one end of the tunnel to the other during
search. There was a slight tendency for bees to make ∪-turns near the
training location, possibly indicating the effect of scent cues. In general,
however, these findings agree with previous results insofar as odometry
appears to play little role in bees' search behaviour in axially striped
tunnels (Srinivasan et al.,
1997).

In all cases where the landmark was present, however, a different pattern
of results emerged. Regardless of whether the landmark was positioned at unit
9 (Fig. 4B), unit 14
(Fig. 4C), or unit 4
(Fig. 4D), bees nearly always
performed first ∪-turns at the site of the landmark (see Appendix for
levels of statistical significance). However, of the flights in which bees
made first ∪-turns at the landmark, not all bees made a second ∪-turn
at this site. Indeed, there was a strong tendency to break visual contact with
the landmark, and in many cases, to fly all the way back to the tunnel
entrance. This tendency to fly a long distance from the landmark is not
unexpected because bees would have been unable to measure distance travelled
relative to the landmark. However, the initial tendency to break visual
contact with the landmark is an unexpected outcome.

Do odometric cues affect landmark fidelity during search?

A comparison was conducted between Experiments 1 and 2 to assess whether
odometric cues can influence bees' fidelity for a landmark cue; that is, the
tendency for bees to remain faithful to the landmark cue throughout the four∪
-turns (Fig. 5). For each
experiment, the three conditions in which the landmark was present were
included in the analysis. Flights in each experimental condition were then
classified according to whether a bee performed its first ∪-turn within
one unit either side of the landmark. The number of flights fitting these
criteria were then divided by the total number of flights, giving a ratio that
measures how strongly bees were attracted to the landmark. Flights showing the
strongest attraction were selected for further analysis, while the others were
excluded from the analysis.

Results of Experiment 2. Bees were trained and tested in an axially striped
tunnel. (A) Bees tested without the landmark generally flew from one tunnel
end to the other. (B-D) Bees tested with the landmark at the training site
(B), shifted to unit 14 (C) and shifted to unit 4 (D), all made first turns
near the landmark then tended to break visual contact on second turns (and
often flew back to the entrance). For an explanation of figure layout and
symbols, see Fig. 2.

Of the remaining flights, the same criteria were applied to the second,
third and fourth ∪-turns, with one additional caveat: only those flights
in which bees had the opportunity to turn within one unit either side of the
landmark were included. For instance, if the landmark was at unit 9, a bee
making its first ∪-turn at unit 8 could not subsequently perform its
second ∪-turn within the set criterion (i.e. one unit either side of unit
9), since a ∪-turn was defined as a crossing from one unit to an adjacent
unit (see Materials and methods). Such flights were therefore also excluded
from further analysis. Taken as a whole, the analysis provides an indication
of bees' affinity to the landmark over the four ∪-turns for each condition
across the two experiments.

To compare these results quantitatively, the data were pooled across all
four ∪-turns and all three conditions within each experiment. The
proportions of bees performing ∪-turns within the set criteria were then
calculated for each experiment and compared statistically. The analysis
revealed a highly significant overall difference between Experiments 1 and 2
(two-tailed Fisher exact test, P<0.001), meaning that the
availability of odometry has a powerful effect on landmark fidelity.
Interestingly, an overall difference (i.e. for all ∪-turns) was also found
between the `Landmark at unit 9' condition, and the remaining two cue-conflict
conditions, within Experiment 1 itself (two-tailed Fisher exact test,
P<0.05), meaning the conflict between landmark position and
odometry decreased bees' overall affinity for the landmark. These fascinating
results are considered in further detail in the Discussion.

Experiment 3

The results of the first experiment suggest that bees use landmark cues at
the feeder to narrow the area of search. However, it is not clear whether the
landmark must be learned during training, or whether bees are intrinsically
drawn towards any landmark in the vicinity of the training location. Indeed,
it is well-known anecdotally that bees are attracted to novel objects in their
environment. How do bees handle a situation in which a novel landmark cue is
added to the training site at test? To examine this issue, bees were trained
to unit 9 in a randomly textured tunnel containing no landmark, and tested
with an unfamiliar landmark in the tunnel. The test protocol was identical to
that used in Experiment 1 (i.e. bees tested in a tunnel in which the feeder
was randomly positioned for short time periods to distribute scent
equally).

Comparison of landmark fidelity, i.e. the tendency for bees to stay
faithful to the landmark during search, with odometry (black line) and without
odometry (grey line). Bees with odometry remained faithful on all four∪
-turns, whereas bees without odometry progressively gave up searching at
the landmark, in all conditions. (A) Landmark in place at the training site,
(B) landmark shifted to unit 14, and (C) landmark shifted to unit 4. Numbers
given indicate the number of bees considered when calculating the observed
frequency.

Fig. 6 shows that the
experimental manipulation was again effective (mean:
F3,173=9.67, P<0.001). Of particular interest
here is the condition in which bees were trained and tested without a landmark
(i.e. `No landmark' condition; Fig.
6A). This condition is comparable to the `No landmark' condition
of Experiment 1, wherein bees were trained with a landmark but tested without.
The question of interest is whether search performance was different in these
two conditions. A statistical comparison of first ∪-turns shows no overall
difference between conditions (Kolmogorov-Smirnov test, P»0.1).
Thus, there is little evidence to support the hypothesis that bees overshoot
the feeder position when trained with a landmark and tested without
(Experiment 1), any more than they would when trained and tested without a
landmark.

The overall pattern of results obtained with the novel landmark in place
was qualitatively different from that seen in Experiment 1. In contrast to
Experiment 1, bees tended to search just short of the landmark (see below for
quantification) in the `Landmark at unit 9'
(Fig. 6B), the `Landmark at
unit 14' (Fig. 6C), and to a
lesser extent in the `Landmark at unit 4' condition
(Fig. 6D). In all three cases,
the search distributions appear truncated near the position of the landmark.
In the `Landmark at unit 4' condition, bees sometimes flew past the landmark
and searched in the tunnel region just beyond the training distance. Since
this consistent tendency to overshoot the training site was unexpected (see
Discussion), more test flights were recorded in this condition than in the
others.

Comparison with Experiment 1

To quantify the apparently different effects of familiar and novel
landmarks, a conjoint analysis was performed on Experiments 1 and 3. Only
first ∪-turns were used for this analysis because they provide information
about the bees' initial reaction to the landmark cue. For each condition in
both experiments, the number of ∪-turns made in the tunnel unit occupied
by the landmark (e.g. unit 9) and the unit just beyond the landmark (e.g. unit
10) was calculated. A second measure counted the number of first ∪-turns
performed in the two tunnel units preceding the landmark (i.e. units 7 and 8).
The ratio of these two numbers (i.e. turns 7,8/turns 9,10) provides a measure
of the tendency for the novel landmark to repel bees rather than attract them.
That is, we interpret ∪-turns performed at units 8 and 9 as resulting from
an attraction effect, while ∪-turns performed at units 7 and 8 are
interpreted as being due to a repulsive effect. By comparing these data across
experiments, it may be possible to deduce the relative behavioural
significance that bees assign to novel and familiar landmarks positioned at
the goal.

The data are plotted in Fig.
7. Black and grey bars show the ratios of bees repelled by the
landmark to those attracted by it for each condition in Experiments 1 and 3,
respectively. The figure shows clearly that, regardless of the landmark's
tunnel location, bees were far more likely to turn just before reaching the
novel landmark than was the case with the familiar landmark. The red line in
the figure indicates the ratios of bees in Experiment 3 that turned in the two
units preceding the landmark relative to all units beyond the landmark (i.e.
not including the landmark unit). This ratio therefore measures the absolute
tendency for bees to be repelled by the novel landmark in each condition of
Experiment 3.

To obtain a quantitative measure of the repulsion effect relative to the
attraction effect (i.e. bars in Fig.
7), the frequencies shown in
Fig. 7 were pooled across all
conditions within each experiment. A statistical test performed on these
overall frequencies shows a highly significant difference between experiments
(two-tailed Fisher exact test, P<0.001). The strength of the
absolute repulsion effect (i.e. within Experiment 3 itself) was ascertained by
comparing the proportions obtained for the `Landmark at unit 4' condition and
the `Landmark at unit 14' condition (i.e. the strongest and weakest effects).
The comparison shows a significant difference in the strength of the effect
(two-tailed Fisher exact test, P<0.05).

Results of Experiment 3. Bees were trained without a landmark and tested
with a landmark. (A) Bees tested without the landmark searched broadly in the
tunnel. (B-D) Bees tested with the landmark (B) at the training site, (C)
shifted to unit 14, and (D) shifted to unit 4, all tended to make first turns
before reaching the landmark. This effect suggests that bees are repelled by
the novel landmark placed along a familiar path. For an explanation of figure
layout and symbols, see Fig.
2.

Assessment of the tendency for a novel landmark to repel bees. Ratios of
bees turning just before the landmark to those turning at the landmark; black
bars, familiar landmark; grey bars, novel landmark. The black line shows the
ratios of bees turning before and after the landmark in Experiment 3 only. The
conditions were `Landmark at unit 4', `Landmark at unit 9' and `Landmark at
unit 14'.

In summary, the behaviour adopted by bees encountering a novel landmark at
the training position is very different from that observed with a familiar
landmark. The novel landmark tends to truncate the search, perhaps because the
presence of an unexpected landmark cue indicates to the bees that they have
overshot the training site and so are in the wrong place (see also
Discussion).

Experiment 4

Do the results of Experiment 1 generalize to longer training distances? In
an attempt to answer this question, bees were trained at unit 21 with a
landmark placed above the feeder. The tunnel was 7.8 m long and lined with a
randomly textured pattern. Bees were tested in the training tunnel under one
of four conditions: `No landmark', `Landmark at unit 21', `Landmark at unit
30' and `Landmark at unit 12'.

Fig. 8 shows that, as in the
previous experiments, landmark position strongly affected search behaviour
(mean: F3,152=79.87, P<0.001). When bees were
tested in the `No landmark' condition (Fig.
8A) there was a tendency for them to search both at the training
location and at a location towards the tunnel end. That is, bees often
overshot the training location considerably. Of the 46 flights in this
condition, bees made their first ∪-turn at or beyond unit 28 (an arbitrary
cut-off) on 21 occasions. On 16 of the 46 flights, the average of the first
two ∪-turns equalled or exceeded 27.

Results of Experiment 4. Bees were trained at unit 21 with a landmark. (A)
Bees tested without the landmark searched either near the training location or
at the end of the tunnel. (B-D) Bees tested with the landmark (B) at the
training site, (C) shifted to unit 30 and (D) shifted to unit 12, all tended
to search at the landmark, or sometimes (as in C) before reaching the
landmark. Note the different y-axis scales in A,C and B,D. For an
explanation of figure layout and symbols, see
Fig. 2.

In the `Landmark at unit 21' condition
(Fig. 8B), the pattern of
search appears very similar to that obtained in Experiment 1 with the landmark
in place at the training location. The distribution is quite narrow and peaks
at the training unit, although there is perhaps slightly more scatter in the
positions of second ∪-turns than in Experiment 1. This increased scatter
is to be expected given that the training distance, and hence odometric error,
was substantially greater in the present experiment. The results of the
`Landmark at unit 30' condition (Fig.
8C) are also quite similar to the analogous condition of
Experiment 1. Indeed, the search distribution appears bimodal, as was the case
in Experiment 1. One peak occurs in the vicinity of the training location,
while the other peak occurs at the position of the landmark. Unfortunately,
the number of flights in this condition was quite low (N=13) due to
inclement weather, which ended the experiment early.

In the `Landmark at unit 12' condition
(Fig. 8D), bees searched
predominantly at the location of the landmark. On only two flights did bees
make first ∪-turns beyond the landmark. In this sense, the results of the
present condition seem to mirror those obtained in the `Landmark at unit 4'
condition of Experiment 1. While the results of the present experiment were
generally comparable with those of Experiment 1, there was one major
difference, manifested in the `No landmark' condition, where bees exhibited a
proclivity to search near the end of the tunnel. However, third and fourth∪
-turn data (not shown above) indicate that bees in the `Landmark at unit
21' and `Landmark at unit 12' conditions also searched at the end of the
tunnel after breaking visual contact with the landmark.

Fig. 9 plots these data for
the third and fourth ∪-turns in the same form used throughout this study
for first and second ∪-turns. It is clear from the figure that, in both
the `Landmark at unit 12' (Fig.
9A) and `Landmark at unit 21' conditions
(Fig. 9B), bees were drawn away
from the landmark, and towards the tunnel end, on third ∪-turns. Bees were
also drawn towards the tunnel entrance (fourth ∪-turns) in the `Landmark
at unit 21' condition. The reason(s) for this seemingly anomalous behaviour
remain unclear (see Discussion). The behaviour did, however, suggest the need
to replicate the present experiment.

Experiment 5

The following experiment was similar to the previous one, in that bees were
trained at unit 21 with the landmark above the feeder. In the control
condition, a separate set of bees was trained to unit 21 without a landmark
and tested under the same conditions. For bees trained with the landmark in
place, there were two test conditions: a `No landmark' condition, and a second
condition in which the landmark was present at the training site (`Landmark at
unit 21'). Bees were again tested in the training tunnel. There were also two
other important differences relative to Experiment 4. The experimental site
was changed to a more open field, due to the overgrowth of trees at the
previous site, and the pattern lining the tunnel walls and floor was changed
from a random texture to a checkerboard. Since the checkerboard pattern is
entirely regular (self-repeating) it mitigates against any tendency for bees
to pinpoint the goal location by matching micro-patterns in randomly textured
tunnels.

Third and fourth ∪-turns, plotted for two conditions of Experiment 4,
show that bees that strayed away from the landmark often searched near the
tunnel end. This occurred with the landmark (A) shifted to unit 12, and (B) in
place at the training location.

The results were in line with the previous experiments in showing an
overall effect for the mean (F2,97=8.61,
P<0.001) search position. Bees in the `Train with and test without
landmark' condition (Fig. 10A)
clearly overshot the training location on first ∪-turns. Indeed, on no
occasion did a bee turn at or before unit 21 (see also below). Interestingly,
the pattern of second ∪-turns shows that bees almost always came back to
the training site on second ∪-turns, unlike the analogous condition of
Experiment 4 where bees often continued to search near the tunnel end. The
results of the `Test with landmark' condition
(Fig. 10B), however, are in
agreement with the first two ∪-turns of bees in Experiment 4, insofar as
bees searched accurately at the landmark, albeit with perhaps a slightly
greater tendency to overshoot the landmark position on first ∪-turns. Nor
was any evidence found that bees behaved radically differently on third and
fourth ∪-turns (data not shown).

The results of the `Train and test without landmark' condition
(Fig. 10C) are particularly
interesting because the pattern of search appears quite different to that
obtained in the `Train with and test without landmark' condition
(Fig. 10A). In particular, it
appears as if the first ∪-turn distribution in the latter condition
(Fig. 10A) was shifted by
about three units beyond the training position, relative to the present
condition (Fig. 10C). No bees
in the `Train with and test without landmark' condition turned at or before
unit 21, whereas the proportion in the present condition was 16 of 39. These
proportions are highly significantly different (two-tailed Fisher exact test,
P<0.001), suggesting that the absence of a familiar landmark can,
in certain circumstances, cause bees to overshoot the training location. In
summary, there was little hint of the anomalous behaviour described in
Experiment 4; namely, the tendency to search at the tunnel end. Although bees
sometimes overshot the training location on first ∪-turns, they almost
always came back to the training site to perform second ∪-turns.

Discussion

The experiments presented herein confirm the hypothesis that familiar
goal-defining landmark cues combine with odometry to ensure that bees search
very accurately at the goal location. These results are therefore broadly
consistent with previous findings on the role of visual landmarks, positioned
en route to the goal location, in the tunnel environment
(Collett et al., 2002;
Srinivasan et al., 1997). The
finding that familiar landmark cues override odometry when the two sources of
information are set in conflict is also consistent with previous studies
conducted under more natural foraging conditions
(Chittka et al., 1995a).

Landmark fidelity and odometric context

Several novel findings also emerged during the course of the study. For
example, it was found that bees were more likely to continue searching near a
familiar landmark, even after initially failing to find food, when odometry
was available (Experiment 1) than when it was absent (Experiment 2). Why were
bees more likely to break visual contact with the landmark when odometric cues
were unavailable? One reason may be that landmark infidelity prevents bees
from searching in the vicinity of a landmark for too long in the absence of an
odometric cue that confirms to the bees that they are in the correct place. In
natural environments, landmark cues at different locations may appear very
similar, and so could easily be confused. One role of odometry then, might be
to distinguish similar-looking landmark cues by acting as a context-setting
cue (e.g. Collett et al.,
1997,
2002), providing bees with
information about the expected location of a landmark.

Results of Experiment 5. Bees were trained at unit 21 with a landmark, and
(A) tested without the landmark, or (B) with the landmark at the training
site, searched in accord with odometry and landmark cues, respectively. (C)
Bees trained without a landmark and tested without a landmark also showed no
signs of the anomalous behaviour observed in Experiment 4. Note the different
y-axis scales in A,C and B. For an explanation of figure layout and
symbols, see Fig. 2.

In the absence of such contextual information, the estimated probability
that a bee is in the wrong place might increase rapidly following the initial
failure to find food, since the landmark has lost much of its power to predict
the presence of food. Thus, a reasonable strategy would be to break visual
contact with the landmark in order to search for similar landmarks nearby.
This would be a particularly useful property in environments where odometric
cues are very sparse, as when bees fly over still water, since it would
prevent bees from persevering with a cue that is itself ambiguous (for the
properties of odometry in differently textured tunnel environments, see
Si et al., 2003). This
property may also suggest a need to revise extant models of landmark guidance
(e.g. Cartwright and Collett,
1983), which do not necessarily capture the flexible interaction
between odometric and landmark cues.

Interestingly, the conflict between odometric and landmark cues, caused by
shifting the landmark in Experiment 1, affected bees differently from the
complete absence of odometry (Experiment 2). In the cue-conflict conditions of
Experiment 1, a small but constant proportion of bees tended to break away
from the immediate vicinity of the landmark, over all four ∪-turns,
relative to the cue-congruent condition. Why should the number of bees
breaking away stay constant? Why should bees not rapidly switch back to
searching in accord with the available odometric information?

Here it may again prove useful to consider that bees can only estimate the
probability that a landmark is in the correct place (i.e. that it is the
correct landmark), since odometric error prevents bees from knowing exactly
where they are at any given time. After the initial failure to find food, this
probability does not change as rapidly as it would in the complete absence of
odometric cues, because odometry provides a contextual cue that is roughly
consistent with the bees being in the correct place. The information available
to the bees therefore favours persevering with the landmark cue. Importantly,
the difference between the cue-congruent and cue-conflict conditions suggests
that the landmark cue did not reset the odometer (e.g.
Chittka et al., 1995b), such
that bees behaved as if they were actually at the training site. That is, why
should bees break away from the landmark on second, third and fourth∪
-turns, when their odometric value has been adjusted to that associated
with the training site? Indeed, the break-away property suggests just the
opposite; that bees do not reset the odometer value, at least relative to the
tunnel entrance. Collett et al.
(2003) reached a similar
conclusion for global path integration in ants.

Landmark repulsion and undershooting

Another significant new finding was that novel landmark cues did not have
the same effect on bees as familiar landmark cues (Experiment 3). Rather than
having an attractive effect, the novel landmark appeared to repel bees
instead, causing them to initiate search closer to the tunnel entrance, than
in the case of the familiar landmark. This finding defines another sense in
which a landmark cue can take behavioural precedence over odometry, and may
reflect another useful behavioural strategy in natural foraging circumstances.
In particular, the presence of a novel landmark on a familiar route might
indicate that the bee has overshot the location of the food source (or, more
generally, that she is in the wrong place). Therefore it may make sense that
bees turn back and begin searching at a shorter distance.

In support of this hypothesis, it was also found that the absolute strength
of the repulsion effect varied with the position of the landmark in the test
tunnel. That is, the closer the landmark was to the tunnel entrance, the
greater the probability of bees making ∪-turns beyond it. This result
makes ecological sense because bees encountering the landmark near the
entrance would (on average) have smaller odometer readings than bees
encountering the landmark at or beyond the training site. Smaller odometer
readings would provide evidence to the bees that they had not overshot the
training site, making them less likely to turn back. Conversely, bees
encountering the novel landmark beyond the training site would (on average)
have larger odometer readings, and so be very likely to turn back.

If this hypothesis were correct, then the subset of bees flying past the
landmark (i.e. ignoring it) in any given condition would (on average) have
smaller odometer readings than bees that turn back. These bees would then tend
to overshoot the training site because they would need to fly a little further
before their odometer readings matched the one stored in memory. This is
exactly what was found in the `Landmark at unit 4' condition of Experiment 3.
The subset of bees that flew past the landmark searched a little beyond the
training distance. This effect appeared so striking during the experiment that
a large number of flights were recorded in that condition in order to confirm
the result. The effect is unlikely to be due to resetting of the odometer at
the novel landmark: additional experiments, not presented here, showed that
bees tended to overshoot the training location even further when the novel
landmark was placed closer to the tunnel entrance. The resetting explanation
would predict the opposite result: namely, bees would be expected to search at
about 9 units beyond the landmark, at a position nearer the tunnel
entrance.

Landmark expectation and overshooting

A third novel finding was that the absence of a familiar goal-defining
landmark can cause bees to overshoot the training site, compared to the case
in which bees were trained without the landmark (Experiment 5; but see below).
In this context, the absence of the expected goal-defining landmark had the
opposite effect of a novel landmark cue. The failure to find the familiar
landmark may have provided evidence to the bees that they had not yet reached
the appropriate distance (i.e. that odometry had brought them up short). Bees
did, however, return to the training site to perform second ∪-turns. This
is an important observation because it shows that bees re-adjusted their
behaviour in response to the failure to find the landmark in the region just
beyond the training site. Thus, bees exhibited two behavioural adjustments in
rapid succession: as an initial adjustment to the absence of the expected
landmark, bees flew a little further than odometry would have permitted; then
as a second adjustment to the failure to find the landmark, bees reverted back
to the learned odometric distance. This result itself exemplifies the amazing
behavioural flexibility of the honeybee.

Interestingly, the overshoot behaviour observed in Experiment 5 did not
arise in the experiments involving the shorter tunnels (Experiments 1 and 3).
The failure to find a positive result may have occurred for several reasons.
For instance, it is possible that bees are likely to exhibit odometric
fidelity at shorter training distances, leading to a diminished effect of the
familiar landmark's absence. Further experiments are required to resolve this
issue.

Why was search often so broad?

Further experiments are also required to assess whether bees behave
differently in indoor and outdoor environments. In particular, the search
performance of bees in some of the present experiments (e.g. Experiments 1 and
3), all of which were conducted outdoors, was considerably less accurate than
would be suggested by the results of comparable experiments conducted indoors
(e.g. Srinivasan et al.,
1997). In general, there are many uncontrollable variables in
outdoor environments (e.g. cloud cover, temperature), which appear to
influence bees' behaviour in the tunnels. Indeed, several experiments had to
be aborted because bees apparently failed to learn, or ignored, the various
cues. One example was included herein, partly to illustrate the point that
bees can sometimes behave anomalously in the test situation (Experiment 4).
Specifically, bees often failed to search in the vicinity of the training
site, and instead searched near the end of the tunnel. This also occurred in
other experiments not included here, and was not clearly related to phototaxis
or any other obvious cue (although bees did often appear to fly quickly in the
tunnel in these experiments, perhaps preventing them from learning the cues
properly; see Chittka et al.,
2003). Since the present work focused on the interactions between
odometric and landmark cues, the results of these experiments were not
included in the paper. Further study is clearly required to understand why
bees can behave differently in indoor and outdoor environments. One
speculative possibility is that conditions in the hive itself may influence
foraging behaviour differently in indoor and outdoor environments
(Groh et al., 2004;
Tautz et al., 2003).

Conclusions

In summary, the present work reveals several strategies employed by bees to
search for food. Bees assess the relative significance of odometric and
landmark cues, often quite dynamically, and assign to each cue a behavioural
weight that is appropriate to the situation. However, further experiments are
required to investigate why bees do not always appear to pay attention to (or
fail to learn) the sensory cues available at the feeder site. Additionally, it
is currently uncertain how the present results might generalise to the scale
of foraging in natural outdoor environments.

Appendix

ANOVAs reported in the main text revealed significant effects of landmark
position on mean search position in all five experiments. Although the ANOVAs
showed that the position of the landmark had a significant effect on where
bees searched, they did not specify exactly which conditions differed. Here we
tabulate the mean search positions, enabling comparison between pairs of
conditions with a simple least significant difference (LSD)
test.⇓

Similar articles

Other journals from The Company of Biologists

Many organizations that use sonar for underwater exploration gradually increase the volume of the noise to avoid startling whales and dolphins, but a new Research Article from Paul Wensveen and colleagues reveals that some humpback whales do not take advantage of the gradual warning to steer clear.

Many animals stabilize their vision by swivelling their eyes to prevent the image from smearing as they move. A new Research Article on tadpoles from Céline Gravot and colleagues shows that contrast between objects in their view affects the strength of this visual reflex, suggesting that the eye may be processing the image at a basic level to produce the reflex.

When starting her own lab at James Cook University, Australia, Jodie Rummer applied for a Travelling Fellowship from JEB to gather data on oxygen consumption rates of coral reef fishes at the Northern Great Barrier Reef. A few years later, Björn Illing, from the Institute for Hydrobiology and Fisheries Science, Germany, followed in Jodie’s footsteps and used a JEB Travelling Fellowship to visit Jodie’s lab. There, he studied the effects of temperature on the survival of larval cinnamon clownfish. Jodie and Björn’s collaboration was so successful that they have written a collaborative paper, and Björn has now returned to continue his research as a post-doc in Jodie’s Lab. Read their story here.

Where could your research take you? The deadline to apply for the current round of Travelling Fellowships is 30 Nov 2017. Apply now!